Regulation of mouse follicle development by follicle-stimulating hormone in a three-dimensional in vitro culture system is dependent on follicle stage and dose

Abstract

The developmental requirements of ovarian follicles are dependent on the maturation stage of the follicle; in particular, elegant studies with genetic models have indicated that FSH is required for antral, but not preantral, follicle growth and maturation. To elucidate further the role of FSH and other regulatory molecules in preantral follicle development, in vitro culture systems are needed. We employed a biomaterials-based approach to follicle culture, in which follicles were encapsulated within matrices that were tailored to the specific developmental needs of the follicle. This three-dimensional system was used to examine the impact of increasing doses of FSH on follicle development for two-layered secondary (100-130 microm; two layers of granulosa cells surrounding the oocyte) and multilayered secondary (150-180 microm, several layers of granulosa cells surrounding the oocyte) follicles isolated from mice. Two-layered secondary follicles were FSH responsive when cultured in alginate-collagen I matrices, exhibiting FSH dose-dependent increases in follicle growth, lactate production, and steroid secretion. Multilayered secondary follicles were FSH dependent, with follicle survival, growth, steroid secretion, metabolism, and oocyte maturation all regulated by FSH. However, doses greater than 25 mIU/ml of FSH negatively impacted multilayered secondary follicle development (reduced follicle survival). The present results indicate that the hormonal and environmental needs of the follicular complex change during the maturation process. The culture system can be adapted to each stage of development, which will be especially critical for translation to human follicles that have a longer developmental period.

FIG. 1. Two-layered secondary follicle growth and metabolism in alginate-collagen I gels

A) Two-layered secondary follicles cultured with increased levels of FSH grew significantly larger. B) The increase in FSH dosage resulted in a significant increase in lactate accumulation on Day 8 of culture. Data represented as average ± SEM, the number examined for each condition is given in Table 1. Points or bars without common superscripts differ significantly between treatments for isolated time points (P < 0.05).

FIG. 2. Two-layered secondary follicle steroid secretion in alginate-collagen I gels

A) Two-layered secondary follicles cultured with 10 or 25 mIU/ml of FSH secreted significantly more progesterone at the end of culture compared to Day 2 (P < 0.05); progesterone levels were not significantly different in response to increased doses of FSH. B) Two-layered secondary follicles cultured with 5, 10, or 25 mIU/ml of FSH secreted significantly more 17β-estradiol at the end of culture compared to Day 2 (P < 0.05). Increased levels of FSH also resulted in a significant increase in 17β-estradiol at the end of the culture. Data are presented as the average ± SEM (n = 3). Points without common superscripts differ significantly between treatments for isolated time points (P < 0.05). Follicles cultured without FSH did not secrete detectable levels of 17β-estradiol or progesterone.

FIG. 3. Oocytes isolated from two-layered secondary follicles cultured in alginate-collagen I gels

A) Oocyte arrested in prophase I from two-layered secondary follicle cultured with 0 mIU/ml of FSH. B) Spindle from a metaphase I-arrested oocyte from two-layered secondary follicle cultured with 25 mIU/ml of FSH. Oocytes were stained with anti-α-tubulin (green) and 4′,6′-diamidino-2-phenylindole (blue). Bar = 10 µm.

FIG. 4. Cross-sections of follicles on Day 8 of culture stained with hematoxylin

Follicles cultured with 0 mIU/ml of FSH (A) or 50 mIU/ml of FSH (C) showed a large number of pyknotic nuclei; this was not observed in follicles cultured with 10 mIU/ml of FSH (B). Arrows indicate pyknotic nuclei. Ooc, Oocyte. Bar = 10 µm.

FIG. 5. Multilayered secondary follicle growth and metabolism in alginate gels

A) Multilayered secondary follicles cultured with increased levels of FSH grew significantly larger. Data represented as average ± SEM, the number examined for each condition is given in Table 1. B) The increase in FSH dosage resulted in a significant increase in lactate accumulation on day 8 of culture. Data are presented as the average ± SEM (n = 3). C) Multilayered secondary follicles cultured with 50 mIU/ml of FSH incorporated more [³H]thymidine from the second to third day of culture. Data are presented as the average ± SEM (n = 5). Points or bars without common superscripts differ significantly between treatments for isolated time points (P < 0.05).

FIG. 6. Multilayered secondary follicle steroid secretion in alginate gels

A) Progesterone was increased significantly at the highest dose of FSH examined. Follicles cultured without FSH did not secrete detectable levels of progesterone. B) 17β-Estradiol increased significantly with time for multilayered secondary follicles cultured with FSH supplemented media. Increased levels of FSH also resulted in significant differences in 17β-estradiol levels. Data are presented as the average ± SEM (n = 3). Points without common superscripts differ significantly between treatments for isolated time points (P < 0.05).

FIG. 7. In vitro-matured oocytes isolated from multilayered secondary follicles cultured in alginate gels

A) Oocytes from follicles cultured in 50 mIU/ml of FSH were not significantly different in size compared to in vitro or in vivo controls. Data represented as average ± SEM, the number examined for each condition is given in Table 2. Points without common superscripts differ significantly between treatments (P < 0.05). B and C) Representative metaphase II spindles from follicles cultured with 5 mIU/ml of FSH (B) or 50 mIU/ml of FSH (C). Oocytes were stained with anti-α-tubulin (green) and 4′,6′-diamidino-2-phenylindole (blue). Bar = 5 µm.